Biology subjects

Balachandar, L.

Publications and source records attributed to Balachandar, L..

5 recordsLinked to original sources

bioRxiv2025

From iPSCs to NPCs to cortical neurons: bioenergetic, neuronal, and calcium signaling phenotypes in bipolar disorder with and without familial mitochondrial disease

BackgroundPsychiatric disorders frequently accompany primary mitochondrial diseases (PMDs), implicating mitochondrial dysfunction as a shared biological substrate for psychiatric vulnerability. To determine how familial mitochondrial risk influences neuronal development in bipolar disorder (BD), we generated induced pluripotent stem cells (iPSCs), neural progenitor cells (NPCs), and cortical neurons (CNs) from three healthy controls (CT), three patients with BD, and three patients with BD and a family history of mitochondrial disease (BD-FMD). MethodsAcross differentiation, we assessed mitochondrial function (ATP production, mitochondrial membrane potential, ROS generation, cytosolic cell-free mtDNA), metabolomic signatures, calcium imaging, and neuronal electrophysiological activity through multi-electrode arrays (MEA). ResultsBD neurons uniquely exhibited pronounced hyperexcitability in comparison to CT and BD-FMD groups. The BD-FMD group displayed prolonged mitochondrial calcium transients, altered membrane potential, and aberrant ROS, in comparison to CT and BD, consistent with a sustained energetic deficit. Metabolomic profiling revealed distinct pathway enrichments in BD and BD-FMD, indicating divergent bioenergetic adaptations to mitochondrial burden. ConclusionsOur findings reveal that familial mitochondrial liability actively reshapes neuronal differentiation and function, producing distinct trajectories of mitochondrial performance, calcium signaling, and network excitability. These results suggest that mitochondrial dysfunction in BD is not a secondary byproduct of illness, but a mechanistic contributor to altered neuronal energetics and communication. Together, these findings delineate how inherited mitochondrial vulnerability reshapes neuronal excitability and metabolism, revealing bioenergetic phenotypes that may inform precision stratification and therapeutic targeting in BD.

neuroscience↗

bioRxiv2025

Investigating the role of cortical microglia in a mouse model of viral infection-induced seizures

Microglia, resident immune sentinels in the brain, are crucial in responding to tissue damage, infection, damage signals like purines (ATP/ ADP), and clearing cellular debris. It is currently unknown how microglial reactivity progresses and contributes to seizure development following Theilers Murine Encephalomyelitis Virus (TMEV) infection. Previously, our group has demonstrated that purinergic signaling in microglia is disrupted in the hippocampus of TMEV-infected mice. However, whether reactive cortical microglia also exhibit changes in purinergic signaling, cytokine levels, and purinergic receptors are unknown. Thus, we seek to evaluate region-based differences in microglial reactivity in the TMEV model. We employed a custom triple transgenic mouse line expressing tdTomato and GCaMP6f under a CX3CR1 Cre promoter and exogenously applied ATP/ADP to acute brain slice preparations from TMEV-infected mice and controls. Interestingly and in contrast to what is observed in hippocampus, we found that despite microglial reactivity in the cortex, microglia can respond to purinergic damage signals and engage calcium signaling pathways, comparable to PBS controls. Using a cytokine panel, we also found that pro-inflammatory cytokine levels (TNF-, IL-1 and IFN-{gamma}) are brain-region dependent in mice infected with TMEV. Using RNAScope-FISH, we observed increases in expression of purinergic receptors responsible for microglial motility (P2Y12R) and inflammation (P2X7R) in the cortex. Collectively our results suggest that following TMEV infection, microglial response to novel damage signals, as well as the production of proinflammatory cytokines, varies as a function of brain region.

neuroscience↗

bioRxiv2024

Reactive microglia fail to respond to environmental damage signals in a viral-induced mouse model of temporal lobe epilepsy

Microglia are highly adaptable innate immune cells that rapidly respond to damage signals in the brain through adoption of a reactive phenotype and production of defensive inflammatory cytokines. Microglia express a distinct transcriptome, encoding receptors that allow them to dynamically respond to pathogens, damage signals, and cellular debris. Expression of one such receptor, the microglia-specific purinergic receptor P2ry12, is known to be downregulated in reactive microglia. Here, we explore the microglial response to purinergic damage signals in reactive microglia in the TMEV mouse model of viral brain infection and temporal lobe epilepsy. Using two-photon calcium imaging in acute hippocampal brain slices, we found that the ability of microglia to detect damage signals, engage calcium signaling pathways, and chemoattract towards laser-induced tissue damage was dramatically reduced during the peak period of seizures, cytokine production, and infection. Using combined RNAscope in situ hybridization and immunohistochemistry, we found that during this same stage of heightened infection and seizures, microglial P2ry12 expression was reduced, while the pro-inflammatory cytokine TNF-a expression was upregulated in microglia, suggesting that the depressed ability of microglia to respond to new damage signals via P2ry12 occurs during the time when local elevated cytokine production contributes to seizure generation following infection. Therefore, changes in microglial purinergic receptors during infection likely limit the ability of reactive microglia to respond to new threats in the CNS and locally contain the scale of the innate immune response in the brain.

neuroscience↗

bioRxiv2021

Serotype-based evaluation of an optogenetic construct in rat cortical astrocytes

Optogenetics is a modern technique which has been recently expanded to non-neuronal cell types, e.g., astrocytes, and involves targeted gene delivery of light-sensitive ion channels like Channelrhodopsin-2 (ChR2). Optogenetic regulation of astrocytic activity can be used for therapeutic intervention of several neurological disorders. Astrocytic gene delivery, viz adeno-associated viral (AAV) vectors, have proven to be robust, time-, and cost-efficient contrary to the generation of transgenic animal models. When transducing astrocytes with an AAV vector, it is imperative to perform a serotype evaluation of the AAV vector due to variability in serotype transduction efficiency depending on species, target region and construct length. Rats have been a very successful animal model for studying a variety of brain disorders, from which ChR2-based intervention of astrocytes will benefit. However, the most efficient AAV capsid serotype targeting astrocytes for ChR2 expression in the in vivo rat brain cortex has not been characterized. To address this, we have evaluated AAV serotypes 1, 5, and 8 of the vector AAV-GFAP-hChR2(H134)-mCherry targeting astrocytes in the rat brain neocortex. Results show that serotype 8 exhibits promising transduction patterns, as it has demonstrated the highest tangential and radial viral spread in the rat brain. Our research will facilitate translational research for future applications of optogenetics involving the transduction of rat brain cortical astrocytes.

neuroscience↗

bioRxiv2019

Unraveling ChR2-driven stochastic Ca2+ dynamics in astrocytes - A call for new interventional paradigms

Control of astrocytes via modulation of Ca2+ oscillations using techniques like optogenetics can prove to be crucial in therapeutic intervention of a variety of neurological disorders. However, a systematic study quantifying the effect of optogenetic stimulation in astrocytes is yet to be performed. Here, we propose a novel stochastic Ca2+dynamics model that incorporates the light sensitive component - channelrhodopsin 2 (ChR2). Utilizing this model, we studied the effect of various pulsed light stimulation paradigms on astrocytes for select variants of ChR2 (wild type, ChETA, and ChRET/TC) in both an individual and a network of cells. Our results exhibited a consistent pattern of Ca2+ activity among individual cells in response to optogenetic stimulation, i.e., showing steady state regimes with increased Ca2+ basal level and Ca2+ spiking probability. Furthermore, we performed a global sensitivity analysis to assess the effect of stochasticity and variation of model parameters on astrocytic Ca2+ dynamics in the presence and absence of light stimulation, respectively. Results indicated that directing variants towards the first open state of the photo-cycle of ChR2 (o1) enhances spiking activity in astrocytes during optical stimulation. Evaluation of the effect of astrocytic ChR2 expression (heterogeneity) on Ca2+ signaling revealed that the optimal stimulation paradigm of a network does not necessarily coincide with that of an individual cell. Simulation for ChETA-incorporated astrocytes suggest that maximal activity of a single cell reduced the spiking probability of the network of astrocytes at higher degrees of ChR2 expression efficiency due to an elevation of basal Ca2+ beyond physiological levels. Collectively, the framework presented in this study provides valuable information for the selection of light stimulation paradigms that elicit optimal astrocytic activity using existing ChR2 constructs, as well as aids in the engineering of future optogenetic constructs. Author summaryOptogenetics - an avant-garde technique involves targeted delivery of light sensitive ion channels to cells. Channelrhodopsin 2 (ChR2), an algal derived light sensitive ion channel has extensively been used in neuroscience to manipulate various cell types in a guided and controlled manner. Despite being predominantly used in neurons, recent advancements have led to the expansion of the application of optogenetics in non-neuronal cell types, like astrocytes. These cells play a key role in various aspects of the central nervous system and alteration of their signaling is associated with various disorders, including epilepsy, stroke and Alzheimers disease. Hence, invaluable information for therapeutic intervention can be obtained from using optogenetics to regulate astrocytic activity in a strategic manner. Here, we propose a novel computational model to assess astrocytic response to optogenetic stimulation which implicitly accounts for the stochastic character of Ca2+ signaling in this cell type. We identified light stimulation paradigms suitable for eliciting astrocytic Ca2+ response within physiological levels in widely-used ChR2 variants and identified highly sensitive parameters in ChR2 kinetics conducive for higher probability in Ca2+ spiking. Overall, the results of this model can be used to boost astrocyte light-induced behavior prediction and the development of improved future optogenetic constructs.

neuroscience↗